CAJ | 학술논문

目的构建心肌Cav1.2通道CT1片段及其突变体与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体,并进行蛋白表达和纯化.方法以豚鼠Cav1.2通道CT1质粒(pGEX?6p?3/CT1)为模板,采用定点突变技术构建CT1 T1603A和CT1 T1603D两种突变体质粒.转化大肠杆菌BL21感受态细胞,大量培养后用IPTG诱导GST融合蛋白表达,分离纯化后采用SDS?PAGE检测CT1及其突变体蛋白的相对分子量和纯度.结果 CT1片段及其突变体融合蛋白得到了正确、大量表达,提取纯化后的CT1片段及其突变体融合蛋白具有较高的纯度.结论成功构建了Cav1.2通道CT1片断及其突变体融合蛋白原核表达载体,获得了CT1突变体融合蛋白,为深入研究CT1片断在Cav1.2通道调节中的作用和机制奠定基础.
목적 구건심기Cav1.2통도CT1편단급기돌변체여곡광감태전이매(GST)중조적융합단백원핵표체재체,병진행단백표체화순화.방법 이돈서Cav1.2통도CT1질립(pGEX?6p?3/CT1)위모판,채용정점돌변기술구건CT1 T1603A화CT1 T1603D량충돌변체질립.전화대장간균BL21감수태세포,대량배양후용IPTG유도GST융합단백표체,분리순화후채용SDS?PAGE검측CT1급기돌변체단백적상대분자량화순도.결과 CT1편단급기돌변체융합단백득도료정학、대량표체,제취순화후적CT1편단급기돌변체융합단백구유교고적순도.결론 성공구건료Cav1.2통도CT1편단급기돌변체융합단백원핵표체재체,획득료CT1돌변체융합단백,위심입연구CT1편단재Cav1.2통도조절중적작용화궤제전정기출.
Objective To construct prokaryotic expression vectors of the CT1 fragment of Cav1.2 channel and its mutants for CT1?GST fusion pro?tein expression and purification. Methods Taking plasmid of pGEX?6p?3/CT1 as template,two mutational plasmids(CT1?T1603A and CT1?T1603D)with site directed mutagenesis were constructed. The plasmids were then transformed to E.coli BL21 competent cells,and the transfor?mants were induced with IPTG for the expression of GST fusion proteins of CT1 fragment and its mutants. SDS?PAGE was performed to determine the relative molecular weight and purity. Results Mutated bases corresponding to the target amino acid site were confirmed by cDNA sequence. High purity of GST?CT1 fusion protein and its mutants were successfully obtained. Conclusion Prokaryotic expression vectors of CT1 fragment and its mutants were constructed,and the fusion proteins were successfully expressed were obtained. These results provided a basis for further studies of the function of CT1 fragment in the regulation for Cav1.2 channel and its mechanism.